Agrobacterium-mediated genetic transformation of Elymus breviaristatus with Pseudomonas pseudoalcaligenes insecticidal protein gene

摘要

An efficient and repeatable protocol for generating transgenic Elymus breviaristatus plants was first established by Agrobacterium-mediated transformation: calli of Elymus breviaristatus were induced from mature seeds, and proliferated on callus induction and maintenance medium containing kinetin 0.05 mg/l and 2, 4-D 8.0 mg/l under dim-light condition (10-20 mu mol/m(2),16 h light) at 26 degrees C. The calli produced were used for transformation mediated by Agrobacterium tumefaciens, EHA105, which carries the binary vector pCAMBIA 1304-ppip, coding for the Pseudomonas pseudoalcaligenes insecticidal protein gene (ppip) and hygromycin phosphotransferase II gene (hptII). The calli transformed were selected and grown in the presence of 60 mg/l hygromycin. Approximately 90 transgenic plants were produced, and transformation frequency of the calli reached 10.52%. The presence of ppip gene in the genomic DNA of regenerated plants was detected by means of PCR and Southern-blot analysis, and the expression of the transgenes was verified by reverse transcription-PCR. Approximately 90% of the tested plants appeared to have only one or two copies of the T-DNA inserts. Verification of anti-grasshoppers activity shows the mortality of grasshoppers reached 16.73%. These results indicated that Agrobacterium-mediated transformation in our condition was effective for transferring foreign genes into Elymus breviaristatus.